SUMMARY Animal behaviors are orchestrated by the sophisticated nervous system, which is dynamically regulated by neuromodulators including lipids and neuropeptides. Endocannabinoids (eCBs) are neurolipids exist broadly in the brain and regulate learning and memory, addiction, pain sensation, and food intake. Among neuropeptides, cholecystokinin (CCK) is involved in nutrient sensing, food intake, and sleep regulation, and oxytocin (OXT) and vasopressin (AVP) play important roles in various aspects of social behaviors. However, how and when lipid and neuropeptide transmission occur in the brain are largely unclear. Existing methods (e.g. microdialysis) that measures brain chemical content suffer from low temporal and spatial resolution. Additionally, since neurolipid and neuropeptide releases often require repetitive neuronal firing and can occur at both axonal and dendritic sites, activity of the neuromodulator- releasing neurons cannot reliably predict where and when neurolipids and neuropeptides are released. Here we propose to develop a set of new tools for long-term monitoring of neurolipids and neuropeptides. Our strategy taps into their natural receptors, human G protein-coupled receptors (GPCRs), which are coupled to GFP. In the presence of neurolipids or neuropeptides, these GPCR Activation-Based (GRAB) sensors transform ligand binding-induced conformational changes into rapid fluorescent signals. We aim to develop and optimize neurolipid and neuropeptide GRAB sensors with >500% fluorescence change (dF/F) and 10- nanomolar affinity in vitro and validate these novel tools in brain slices ex vivo and mouse behavioral paradigms in vivo. In Aim 1, we will develop GRAB sensors for endocannabinoids, CCK, vasopressin, and OXT by systematically varying key sites involved in ligand binding, conformational change, etc. In Aim 2, we will validate the performance of these sensors in brain slice following long-term expression using viral tools. In Aim 3, we will use three different imaging methods (fiber photometry, epifluorescence and 2-photon imaging coupled with GRIN lens) in different behavioral paradigms to test in vivo performance of the novel GRAB sensors in mice. Feedback from experiments in Aims 2-3 will guide iterative optimization in Aim 1. Successful completion of our proposal will yield a suite of powerful tools and technical approaches, which will greatly facilitate studies of neurolipids and neuropeptides under both physiological and pathological conditions, helping reveal disease mechanisms, providing therapeutic guidance, and eventually benefiting human health.